The subject of this proposal is our work with the Hepatitis B virus (HBV) transcriptional enhancer region. HBV is one of the few viruses that is clearly linked with the genesis of human cancer, specifically hepatocellular carcinoma. Little is known about the regulation of viral gene expression. The research in my laboratory is focused on the study of eukaryotic enhancer elements, sequences that activate transcription of a gene in an orientation and location independent fashion. The hepatotropic phenotype of HBV gene expression is conferred, in part, by the viral enhancer region. The proposed studies will provide important information concerning the regulation of HBV gene expression. Further, these analyses will provide information concerning general transcriptional control mechanisms and liver-specific gene expression. The specific goals of this proposal are the following. We have identified a nuclear protein, termed EF-C, that binds to the HBV enhancer region. The EF-C site is a functional component of several viral enhancers. I propose to mutate the ER-C site, using site-specific point mutagenesis, and analyze the role of this element in the regulation of several HBV promoter regions using a transient expression assay in liver cells and in the context of an intact, replication virus. We have demonstrated that at least two other elements in the HBV enhancer region function in concert with the EF-C site to enhance transcription. These elements appear to act synergistically and with spacial constraints with respect to one another. We will precisely define these functional elements within the HBV enhancer genetically, and analyze the activity of these elements in liver and non-liver cell lines. I propose to identify and purify the specific regulatory proteins that bind to these domains, and establish a functional correlation between DNA=protein binding and enhancer region activity. The possible cooperative binding of the identified proteins with EF-C will be activator, augments the activity of the HBV enhancer through a sequence-specific mechanism.